Francisella tularensis (Ft) causes tularemia, a serious and potentially fatal disease. Because Ft has an extraordinarily high infectivity, causes high morbidity and mortality, is relatively easily cultured and dispersed, and has previously been weaponized, it is classified as a Category A potential agent of bioterrorism. As post- exposure prophylaxis is not a practical public health alternative for countering an outbreak of pneumonic tularemia, a safe and effective pre-exposure vaccine is needed. The goal of this application is a safer and more potent vaccine against aerosolized Ft than the current unlicensed, toxic, and insufficiently effective vaccine (LVS). Our strategy is to utilize a live attenuated recombinant homologous vector - an attenuated form of the LVS vaccine (this parent vaccine has already been tested in humans) - to overexpress highly immunoprotective Ft proteins such as IglC, previously demonstrated in this laboratory to enhance protective immunity against aerosolized Ft. The proposed 2nd generation recombinant LVS (rLVScapB) vaccine will be safer than LVS because the proposed vector already has been demonstrated to be >10,000 times more attenuated than LVS and yet induce strong cell-mediated and humoral immune responses. The proposed 2nd generation rLVScapB vaccine will be more potent than LVS because it will overexpress large amounts of highly immunoprotective Ft proteins via novel promoters. This strategy for a tularemia vaccine mimics that used successfully in this laboratory to develop the first vaccines against tuberculosis that are safer and more potent than the current BCG vaccine; one of these tuberculosis vaccines has already demonstrated safety and enhanced immunogenicity in human trials. In this application, to accomplish our goal of a vaccine against tularemia that is safer and more potent than LVS, we propose to build upon our preliminary success with 1st generation rLVScapB vaccines by constructing the aforementioned new 2nd generation rLVScapB vaccines and testing them systematically for stability; protein expression extracellularly and intracellularly; safety; immunogenicity; and near-term and long- term efficacy against lethal Ft challenge in a mouse model. At the same time we shall determine optimal mucosal and systemic routes for administration. By the completion of this project, we anticipate having a vaccine that is substantially safer and more potent than LVS and suitable for testing in humans. While the focus of our grant is on a tularemia vaccine, our approach is applicable to vaccines against intracellular pathogens in general. Thus, lessons learned and strategies developed during the development of a successful tularemia vaccine are likely to be broadly applicable.